Triggering of Ca2+ signaling though CRAC (Ca2+-release-activated Ca2+) channels by T cell receptor (TCR) stimulation is a key step for T cell functions. The PI has recently identified and named Orai1 as a long-sought pore component of the CRAC channel. We has shown that human patients with a missense mutation in the Orai1 gene have lethal, severe combined immune deficiency (SCID) emphasizing its predominant role in the function of immune cells. In addition, analyses of Orai1-null mice have shown that Orai1 is critical for B, T, and mast cell activation. Identification of Orai1 provides a molecular target to develop small molecules that can block immune responses. Such small molecules can be potentially used as immunosuppressants to alleviate immune complications during transplantation or autoimmune disorders. Our mutational analysis of Orai1 identified such a molecule while characterizing a novel inactivation mechanism that involves the Orai1 intracellular loop located between transmembrane (TM) segments II and III. Mutation of the loop residues N153VHNL157 flanked by two prolines, removed channel inactivation. Experiments carried out with concatenated Orai1 tetramer indicate that CRAC channels are strongly inhibited by one of four intracellular loops, suggesting dominant block by a single loop. Finally, exogenous expression of a peptide derived from the TM II-TM III loop blocked CRAC channels in T cells. The strong dominant blocking effect of the inactivation particle on the CRAC channel activity will definitely benefit development of the peptide as an immunosuppressant. In the current proposal, we will investigate whether the inhibitory peptide may be used as an immunosuppressant. The specific aims are as follows: (1) To determine the structural requirement of the inhibitory peptide derived from the Orai1 inactivation loop. We will determine the minimum size of the peptide required to block the CRAC channel activity. A short protein transduction sequence will be attached onto the blocking peptide to generate a membrane-permeant form and its blocking effect on the T cell function will be tested. (2) To test the possibility that the short inhibitory peptide of Orai1 can be used as immunosuppressant in a clinically relevant animal model. First, we will determine the blocking efficacy of the peptide in cytokine production and proliferation of primary immune cells. Then, we will determine the effect of the inhibitory peptide in the onset/progress of a mouse graft-versus- host disease model. Development of a CRAC channel inhibitory peptide would represent a novel and improved therapeutic approach to suppress immune responses. Such a peptide is likely to have much less side effects than cyclosporin A and tacrolimus since the inhibitory peptide is specifically targeting the CRAC channel activity that is predominant mostly in immune cells. PUBLIC HEALTH RELEVANCE: We propose to develop a peptide blocker that can suppress calcium entry in immune cells, thereby immune responses. Our structure/function analysis of a newly described component of the CRAC channel, Orai1 identified a novel peptide that can inactivate calcium entry specifically triggered by immune receptor stimulation, and the possibility of development of the blocking immune activation will be tested using an animal model of graft-versus-host disease. Our study can benefit development of drugs that can repress immune functions as therapy for immune system-related problems such as graft rejection or autoimmune disease.